One characteristic that contributes to Enterococcus faecalis[unreadable]s success as the second or third leading cause of hospital-acquired infection in the U.S. is its flexible metabolism, which enables survival under a wide variety of conditions. However, little is known about how E. faecalis regulates gene expression involved in employing alternative energy sources. The objective of this application is to elucidate the post-transcriptional mechanisms involved in regulating ethanolamine utilization, a potentially valuable source of carbon and nitrogen in the host. Our central hypothesis is that an activated two-component system (TCS) and adenosylcobalamin (AdoCbl) bind to specific regions in the untranslated RNA (UTR) of the ethanolamine utilization operon (eut) operon proximal to predicted terminators preventing their formation. The response regulator of the TCS, EutV, is a member of the AmiR and NasR Transcriptional Antiterminator Regulators (ANTAR) that have RNA-binding activity. The rationale for this research is that it will increase fundamental understanding of RNA-based gene expression control and such knowledge will contribute to efforts focused on exploiting regulatory RNAs as drug targets. Several features of the post-transcriptional regulatory control elements of this operon implicate an expanded mechanistic diversity, well beyond the current thinking. Aim #1 will elucidate the transcriptional organization of the eut operon. We postulate that specific features contained in the operon function to tightly modulate the levels of expression of the many genes in the locus. We will experimentally test our hypothesis by identifying the transcripts, transcriptional start sites, and gene expression levels of the eut operon. Additionally a bioinformatic analysis of loci regulated by ANTARs will be conducted to identify conserved features. Aim #2 will identify the components and mechanism of action of the E. faecalis Eut antitermination complex as a general model for antitermination by ANTARs. We postulate that ethanolamine-induced autophosphorylation and phosphorylation of EutW and EutV respectively causes the formation of a complex with antiterminator activity. A variety of in vivo and in vitro approaches coupled with mutational analysis will be performed to prove the hypothesis. Finally, Aim #3 will determine the role of the AdoCbl riboswitch and the TCS on gene regulation at the second terminator in the eut operon where both are postulated to be required to fully destabilize the terminator, again using a variety of in vivo and in vitro approaches. The studies proposed are significant and innovative because they will elucidate what are currently poorly understood post-transcriptional regulatory mechanisms, contributing groundbreaking knowledge to the field of gene regulation and possibly to the development of therapeutics that target regulatory RNAs.